Protein s nitrosylation and its relevance to redox control of cell signaling

CONCENTRATION OF H 2 O 2 CAUSES NON-SNO OXIDATIVE 76 3.5.1 Intracellular NO˙ is decreased with an increase in O2˙- generation whereas it is actively synthesized by H2O2 and growth facto

PROTEIN S-NITROSYLATION AND ITS RELEVANCE TO REDOX CONTROL OF

CELL SIGNALING

KYAW HTET HLAING (M.B.B.S, UM 2)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2012

DECLARATION

I hereby declare that this thesis is my original work and it has

been written by me in its entirety I have duly acknowledged all the sources of information which have been

used in the thesis

This thesis has also not been submitted for any degree in any university previously

Kyaw Htet Hlaing

24 Dec 2012

Acknowledgements

I wish to express my deepest gratitude to my supervisor, Associate Professor Marie-Véronique Clément, Department of Biochemistry, for introducing me into the field of “Redox Control of Cell Signaling”, and guiding me along the arduous journey

of my Ph.D study I am truly grateful for her warm encouragement and constant optimism in the face of “reality of day-to-day life of a graduate student” over the years This thesis has not been complete without her unending support and kind understanding I also like to thank my TAC members, Dr Andrew Jenner and Professor Kini R Manjunatha, for their comments, useful advice and feedbacks throughout my study

My heart-felt thanks to my lab members for listening to both of my happy and frustrating stories Spending time together with them has made my life in the lab most enjoyable I want to thank Luo Le in particular for taking time to read the draft of my thesis and giving me useful feedback Also my special thank to Ms Lee Mui Khin for keeping things in order and making sure that I always get what I need in time

Lastly, my deepest gratitude to my family for their encouragement and support all along I wish to express my special thank to my older sister, Ms Wint Wint Htet Hlaing, for helping me out financially when in need and motivating me when confronted with various setbacks during my study

1.2.2 Hydrogen Peroxide and Hydroxyl Radical 6 1.2.3 Nitric Oxide and its derivatives 6 1.3 EFFECTS OF REACTIVE OXYGEN AND NITROGEN SPECIES ON

1.3.2 Physiological Function: Redox Signaling 10 1.4 MECHANISMS OF REDOX-BASED REGULATION OF CELL SIGNALING: FUNCTIONAL CONSEQUENCES OF OXIDATION OF

1.4.3 Multimerization of Subunits 17

1.4.5 Oxidation of Transcription Factors 18

1.8.1 Factors influencing protein S-nitrosylation 25

2.2.2 Sodium Dodecyl sulphate polyacrylamide gel electrophoresis

2.2.5 Detection of S-nitrsoylated and Oxidized PTEN by

2.2.6.1 Detection of Total Protein and PTEN S-nitrosylation 47 2.2.6.2 Detection of Total Protein and PTEN Oxidation 48

2.2.7 Lucigenin Chemiluminiscence Assay for Detection of Intracellular

3.1.4 Both pharmacological inhibition and siRNA gene silencing of

Cu-Zn SOD induce protein S-nitrosylation 64

CONCENTRATION OF H 2 O 2 CAUSES NON-SNO OXIDATIVE

76

3.5.1 Intracellular NO˙ is decreased with an increase in O2˙- generation whereas it is actively synthesized by H2O2 and growth factors 88

3.5.2 Identification of S-nitrosylation species for oxidants- and growth

3.5.3 Peroxynitrite: oxidation vs nitration 99

3.5.4 Role of calcium in protein S-nitrosylation caused by ROS and

3.5.5 GSNOR inhibition enhances protein S-nitrosylation 107

3.5.6 Inhibition of O2˙- production enhances protein S-nitrosylation through

3.6.1 Scavenging PNOO˙ prevents PDGF activation of Akt kinase whereas

3.6.2 O2˙-/ NO˙ Balance in Signal Transduction 115

3.6.3 ONOO- mediates Akt activation by O2˙- and low concentration of

3.7.1 Maintenance of protein S-nitrosylation in the absence of serum is

associated with sustained signal transduction in precancerous and cancer

3.7.2 Protein de-nitrosylation in cancer 126

129

OXIDATION USED BY O 2˙- AND PHYSIOLOGICALLY RELEVANT

4.1.3 Redox Signaling: O2˙- vs H2O2 131

4.2.1 PTEN: an example of oxidative modification of protein upon

growth factor induction of cell proliferation 133

4.4 O 2 - AND NO˙: STRIKING THE RIGHT BALANCE FOR SIGNAL

Summary

Discovery of the function of oxidants as signaling molecules marks the beginning of the field of redox control of cell signaling Understanding the mechanism of how free radicals regulate signaling is critical to distinguish between normal physiology and cellular toxicity both caused by reactive species It is now known that free radicals influence various cellular processes by altering the function

of critical proteins as a result of reversible oxidation of “reactive cysteine” within the proteins Different types of oxidative modification such as S-nitrosylation, S-glutathionylation, di-sulphide bond formation, sulphenic acid formation, have been proposed to mediate redox control of cell signaling However, physiological relevance

of these modifications is somehow missing Furthermore, there has been a debate about relative importance of O2˙- versus H2O2 in mediating enhanced cell proliferation Following up on our previous study that demonstrates that O2˙- activates survival kinase Akt through S-nitrosylation of the tumor suppressor PTEN, our current study deciphers the mechanistic aspect of how oxidative signal by O2˙- is transformed into nitrosative signal We also provide evidence that physiologically relevant concentration of H2O2 predominately induces protein S-nitrosylation over non-SNO modifications We demonstrate that protein S-nitrosylation induced by O2˙-

and H2O2 is both mediated by common S-nitrosylating species, ONOO- although the pathways to formation of ONOO- are different in each case

Moreover, we show that oxidation of proteins that occurs following incubation with PDGF, EGF and 10% FBS is by protein S-nitrosylation Particularly in the case

of PDGF, the growth factor does not generate a high level intracellular H2O2

regardless of concentration of PDGF used and it consistently induces protein

S-nitrosylation Again, we find that the relevant S-nitrosylating species that mediates growth factors-induced protein S-nitrosylation is ONOO- Removal of ONOO-prevents protein S-nitrosylation as well as activation of Akt induced by O2˙-, H2O2 and PDGF demonstrating protein S-nitrosylation is of relevance to redox control of cell signaling

We also highlight the consequences of disturbing O2˙-/NO˙ balance in cell signaling On one hand, removal of NO˙ is effective in preventing S-nitrosylation but

it increases the levels of intracellular O2˙- and H2O2 potentially causing oxidative stress with damaging consequences On the other hand, we demonstrate the ineffectiveness of removing O2˙- alone to stop pro-survival signaling as the latter could continue by ONOO--independent but NO˙-dependent S-nitrosylation

Lastly, we show that increased ROS and RNS production in breast cancer cell line (MCF7) correlate with sustained protein S-nitrosylation and Akt activation in the absence of serum However, the prevalence of this finding still has to be tested in other types of cancers We also find that protein S-nitrosylation and Akt activation in MCF7 is very stable requiring further studies on identifying the factors contributing to this stability

List of Figures Figure 1: NOX2 (gp91phox) activation and generation of O2˙- in phagocytes 5

Figure 2: Consequences of oxidation of reactive cysteines within the target proteins

15 Figure 3: Schematic representation of various types of reversible cysteine oxidations

20

Figure 5: Compartmentalization of cellular NO˙ source and its targets 27

Figure 6: Mechanism of enzyme-mediated protein de-nitrosylation 30

Figure 7: Serum withdrawal results in a decrease in base level O2˙- production 53

Figure 8: Inhibition of Cu-Zn SOD by 1mM DDC caused an increase in intracellular

O2˙- production but a decrease in intracellular H2O2 56

Figure 11: Both NO˙ donor and trannitrosylating agent induce protein

S-nitrosylation in mouse embryonic fibroblasts 62

Figure 12: Pharmacological inhibition of Cu-Zn SOD induces protein S-nitrosylation

65

Figure 13: siRNA gene silencing of Cu-Zn SOD led to an increase O2˙- in

production, a decrease in H2O2 level and induction of protein S-nitrosylation 66

Figure 14: Exogenous H2O2 treatment increases intracellular H2O2 level but has no

Figure 15: Protein S-nitrosylation occurs predominately at low concentrations of

H2O2 although H2O2 causes protein oxidation in a dose dependent manner 72

Figure 16: High concentration of H2O2 is toxic to the cells 74

Figure 17: 50uM H2O2 induces protein S-nitrosylation in a time dependent manner

75

Figure 18: ROS production during growth factors signaling 78

Figure 19: Growth factors induced protein S-nitrosylation 80

Figure 20: Protein S-nitrosylation is maintained in the presence of high concentration

Figure 21: Slow release NO˙ donor, Deta-NONOate and tranS-nitrosylating agent,

Figure 22: Exposure of cells to O2˙-, H2O2 and PDGF all S-nitrosylate tumor suppressor, PTEN in a time dependent manner 85

Figure 23: S-nitrosylation of PTEN predominately occurs at low concentrations of

H2O2 while it is equally induced by all concentrations of PDGF treatment 88

Figure 24: Increase in intracellular O2˙- is associated with decrease in intracellular NO˙ level whereas exogenous H2O2 treatments cause increased production of

Figure 25: PDGF, EGF and 10% FBS all increase intracellular NO˙ 91

Figure 26: Intracellular NO˙ is essential for protein S-nitrsylation induced by O2˙-,

Figure 27: Total protein and PTEN S-nitrosylation induced by O2˙-, low concentration H2O2 and PDGF may depend on formation of peroxynitrite 97

Figure 28: L-NMMA reduces basal production of NO˙ in MEF cells and prevents

new production of NO˙ stimulated by low concentration of H2O2 and PDGF 98

Figure 29: Low concentrations of exogenous ONOO- induce total protein and PTEN

S-nitrosylation whereas at high concentration, it causes non-SNO oxidative

Figure 30: 50uM H2O2 causes an increase in intracellular Ca2+ whereas 10ng

PDGF has on effect on intracellular Ca2+level 104

Figure 31: Intracellular release of Ca2+enhances protein S-nitrosylation through

Figure 32: Enhancement of protein S-nitrosylation by GSNOR inhibition is through

Figure 33 A GSNOR inhibitor, C3 enhances total protein and PTEN snitrosylation

Figure 34: Inhibition of O2˙- generation enhances protein S-nitrosylation 112

Figure 35: Scavenging ONOO- prevents Akt-phosphorylation by PDGF whereas

Figure 36: NO˙ scavenging increases intracellular ROS level that maintains Akt

Figure 37: Inhibition of O2˙- alone does not affect Akt activation by PDGF but

Figure 38: ONOO- attenuates Akt activation by O2˙- and low concentration of H2O2

120

Figure 39: Protein S-nitrosylation is maintained in the absence of serum in MEF

Figure 40: Increased S-nitrosylation in MCF7 breast cancer cells 124

Figure 41: Akt phosphorylation is maintained in the absence of serum in MEF K/O

Figure 42: FeTPPS de-nitrosylate proteins and dephosphorylate Akt in MEF WT but

Figure 43: Proposed pathway for the formation of ONOO- upon an increase in

Figure 44: Low concentration of H2O2 decrease intracellular Cu-Zn SOD activity 137

Figure 45: Proposed pathway of ONOO- formation by exogenous H2O2 138

Figure 46: Proposed pathway for ONOO- formation by growth factors 140

Figure 47: The interplay of NO˙, O2˙-, ONOO-, and NO2- 141

Figure 48: Proposed pathways for ONOO- formation and S-nitrosylation 144

Figure 49: Schematic representation of the impact of O2˙- and NO˙ balance in cell

List of Tables Table 1: Reactive Oxygen Speciess and Reactive Nitrogen Speciess 2

Table 3: Major Reactive Nitrogen Speciess in Biological System 8

Table 5: Enzymes that reduce reversible cysteine oxidation 20

Abbreviations

Akt Protein kinase B

Ang II Angiotensin II

Biotin-HPDP N-6-Biotinamido-hexyl-3ʹ′-2ʹ′-Pyridyldithio-Propionamide c-PTIO 2-4-Carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-

oxide potassium salt Cu-Zn SOD Copper Zinc Superoxide dismutase

DMSO Dimethylsulfoxide

DPI Diphenyleneiodonium chloride

DTT Dithiothreitol

EDTA Ethylenediamine tetraacetic acid

EGF Epidermal growth factor

FBS Fetal Bovine Serum

FeTPPS 5,10,15,20-Tetrakis (4-sulfonatophenyl)prophyrinato iron (III),

chloride GSH Reduced glutathione

GSSG Glutathione disulfide

Hepes 4-(-2-hydroxyethyl)-1- piperazineethanesulfonic acid

H2O2 Hydrogen peroxide

KO cell MEF PTEN-/-cell

L-NMMA NG – monomethyl – L – Arginine Monoacetate

MAPK Mitogen-activated protein kinases xiv

MEF Mouse embryonic fibroblast

NOS Nitric Oxide Synthase

N2O3 Dinitrogen trioxide

OH˙ Hydroxy radical

ONOO - Peroxynitrite

PBS Phosphate buffered saline

PDGF Patelet-derived growth factor

PIP3 Phosphatidylinositol-3,4,5-trisphosphate

PI3-K Phosphatidylinositol 3 - kinase

PP2A Protein phosphatase 2A

PTEN Phosphatase and Tensin Homolog Deleted on Chromosome 10

PTP Protein tyrosine phosphatase RBS Reactive bromine species

RCS Reactive chlorine species

RNS Reactive nitrogen species

ROS Reactive oxygen species

RSS Reactive sulphur species

VEGF Vascular endothelial growth factor

3-NT 3-nitrotyrosine

CHAPTER 1: INTRODUCTION

The history of free radicals dates back to the time when oxygen was first recognized as a toxic gas in 1954 (Gershman R et al, 1954) Initially, it was suggested that toxic properties of oxygen could come from its direct inhibition of essential enzymes (Hauggard N, 1968), but subsequent findings revealed that these damaging effects are rather due to the action of oxygen-derived radicals (Glibert DL 1981)

A free radical can be defined as any species capable of independent existence that contains one or more unpaired electrons in atomic or molecular orbits (Halliwell

B and Gutteridge JMC, 2007) It is this unpaired electron(s) that make(s) free radicals highly reactive, but the degree of reactivity varies widely among different radicals Not all free radicals derive from molecular oxygen There are many other types of non-oxygen derived free radicals made in living systems, namely; carbon-centre radicals such as CCl3˙, most transition metal ions with exception of zinc and some

oxides of nitrogen such as NO˙ and NO2 (Halliwell B and Gutteridge JMC, 2007)

Current nomenclature of reactive species includes reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive chlorine species (RCS), reactive bromine species (RBS) and reactive sulphur species (RSS) Some reactive species belong to more than one category, for example, hydrobromous acid, HOBr is considered both as ROS and RBS, and peroxnitrite, ONOO- is referred to as both ROS and RNS Also note that “reactive species” is a collective term and they could either be radicals or non-radicals that are oxidizing agents easily convertible to

radicals (Halliwell B and Gutteridge JMC, 2007) Among reactive species, ROS and RNS have the widest range of biological functions and they are the main subjects of discussion throughout this thesis Table 1 shows the list of ROS and RNS that are biologically important in living organisms

Table 1: Reactive Oxygen Species and Reactive Nitrogen Species

Reactive oxygen species Reactive Nitrogen Species Radicals Nonradicals Radicals Nonradicals

Superoxide (O 2 ˙-), Hydrogen peroxide (H 2 O 2 ), Nitric oxide (NO˙) Nitrous acid (HNO 2 ),dinitrogen trioxide/tetroxide

hydroxyl (OH˙), peroxyl, hypochlorous acid (HOCl), nitrogen dioxide (N2O3/N2O4), nitronium ion (NO2+), peroxynitrite (RO 2˙),alkoxyl (RO˙), ozone (O3), singlet oxygen (NO˙2 ) (ONOO-), alkyl peroxynitrite (ROONO), hydroperoxyl (HO2˙) (1

ΔgO 2 ), peroxynitrite nitroxyl anion (NO - ), nitrosyl cation (NO+), (ONOO - ) nitryl chloride (NO2Cl)

(Adapted from Rigas B and Sun Y, 2008)

Reactive species are generated during irradiation by UV light, by X-rays and

by gamma-rays or exist as pollutants in the atmosphere In the biological systems, ROS are produced as by-products of mitochondria-catalyzed electron transport reactions or intentionally generated by neutrophils and macrophages during innate immunity (Cadenas E, 1989; Halliwell B and Gutteridge JMC, 2007)

1.2.1 Superoxide

The first byproduct of aerobic metabolism within mitochondria is superoxide (O2˙-) During the process of oxidative phosphorylation, a small number of electrons leak from the mitochondrial transport chain to oxygen prematurely, forming the oxygen free radical O2˙- This leakage occurs mainly at complexes I and III (Cadenas

E and Davies KJ, 2000) Another important source of O2˙- production is by induced activation of membrane-bound enzyme systems such as the NADPH oxidase complex (NOX) Superoxide generation by the NOX complex is deliberate and it was best characterized in phagocytic cells such as neutrophils that undergo a series of reactions called the respiratory burst in response to microorganisms or inflammatory mediators (Babio BM et al, 2002) The enzyme complex consists of six subunits- two membrane-bound components, p91phox, p22phox which together form cytochrome b558, the enzymatic centre of the complex, and four cytosolic proteins, p47phox, p67phox, p40phox and the small guanosine triphosphate (GTP)-binding protein Rac1 and Rac2 This enzyme system was the first to disprove the rule that O2˙- was generated accidentally and served no particular cellular function During the 1990s, the similar enzyme complex systems were found in various tissues other than phagocytes accounting for non-mitochondrial source of O2˙- production (Banfi B et al, 2003; Cheng G et al, 2001; De Deken X et al, 2000; Edens WA et al, 2001; Geiszt M

stimulus-et al, 2000 & 2003; Lambstimulus-eth JD stimulus-et al, 2000) There are seven isoforms identified so far but the other six isoforms produce O2˙- at a fraction (1-10%) of the level produced

in neutrophils by NOX2 (Lambeth JD, 2004 & 2007; Petry A et al, 2010) Tissue distribution of NADPH oxidase isoforms and their known regulators are summarized

in the following table:

Table 2: Human NOX/DUOX enzymes

(Adapted from Lambeth JD, 2004)

NOX isoforms are homologues of gp91phox subunit that accounts for ROS generation The regulation of gp91phox (NOX2) is well characterized (Groemping Y

et al, 2003; Huang and Kleinberg, 1999; Vignais PV, 2002) but little is known about the regulation of other isoforms Generally, the catalytic component of NOXs responsible for generation O2˙- resides within the membrane structure whereas regulatory subunits scatter in the cytosol Upon activation, cytosolic components are recruited to the membrane and form a mutually stabilizing complex with membrane catalytic subunits The sequence of events leading to full activation of NOX is given for the prototypic isoform NOX2 in Figure 1

(Adapted from Lambeth, 2004)

Figure 1: NOX2 (gp91phox) activation and generation of O 2 ˙ - in phagocytes

At least three signaling cascades mediate the activation process First, PI3K provides lipid docks for p40phox and p47phox to station in the membrane Second, phosphorylation of p47phox by protein kinases such as PKC and Akt promotes its binding to p22phox by relieving autoinhibition in p47phox Third, activation of guanine-nucleotide exchange results

in active Rac-GTP, which then binds to p67phox for complete assembly of the holo enzyme for generation of O 2 ˙-

Other intracellular sources of O2˙- generation include xanthine oxidase (Fridovich I, 1970), NADPH cytochrome p450, lipoxygenase and cyclooxygenase (Goeptar AR et al, 1995) and the uncoupled nitric oxide synthase (Alderton WK et al, 2001) However O2˙- generated from theses sources is associated with various diseased conditions such as hypertension and diabetes (Dixon LJ et al, 2003 & 2005; Jankov RP et al, 2008)

1.2.2 Hydrogen Peroxide and Hydroxyl Radical

Hydrogen peroxide (H2O2) is produced directly in cells by several enzymes such as glucose oxidase (Bankar SB et al, 2009), xanthine oxidase (Kelley EE et al, 2010) DUOX1, DUOX2 (Edens WA et al, 2001; Donkó A et al, 2005), and peroxisomes (Fritz R et al, 2007) And it is also derived from two molecules of O2˙- in

a reaction called dismutation, which is accelerated by the enzyme, superoxide dismutase (SOD)

O2˙- + O2˙- + 2H+ → H2O2 + O2

H2O2 interacts with O2˙- to generate highly reactive hydroxyl radical (OH˙) by the iron-catalyzed Haber-Weiss reaction as follows:

Fe3+ + O2˙- → Fe2+ + O2 (1) The second step is the Fenton reaction:

Fe2+ + H2O2 → Fe3+ + − + OH˙ (2)

Net reaction:

O2˙- + H2O2 → OH− + OH˙ + O2 (3)

In phagocytes, the enzyme myeloperoxidase produces HOCl from H2O2 (Anderson

MM et al, 1999), which contributes to the inflammation of tissues during immune defense response

1.2.3 Nitric Oxide and its derivatives

Nitric oxide (NO˙) is a colorless gas that contains an unpaired electron on the anti-bonding 2π orbital, and thus is a radical Since it is soluble in organic solvents, NO˙ can cross membranes and diffuse readily NO˙ reacts slowly with most biological molecules The removal of the unpaired electron results in nitrosonium cation, NO+

whereas one-electron reduction gives nitroxyl anion, NO– Both derivatives are more reactive than the parent NO˙ molecule (Stamler JS et al, 1992)

NO˙ is synthesized in biological tissues by the nitric oxide synthese (NOS) enzymes, which metabolize arginine to citrulline with the formation of NO˙ via five electrons oxidative reaction (Andrew PJ and Mayer B, 1999; Ortiz de Montellano PR

et al, 1998) Synthesis of endogenous NO˙ is highly regulated by the activity of isoforms of nitric oxide synthase (NOS) There are three types of NOS Neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3) are constitutively expressed in nervous system tissues and endothelia cells respectively (Bredt DS et al, 1990; Knowles RG et al, 1989; Palmer LA et al, 1988) Inducible NOS (iNOS or NOS2) was first identified in phagocytes in response to endotoxin or cytokines (Billiar et al, 1990; Marletta MA et al, 1988; McCall TB et al, 1989) While eNOS and nNOS require Ca2+ for their activation, iNOS enzymes are Ca2+ independent and upon induction, iNOS can generate highly localized concentration of NO˙ up to the micromolar range (Alderton W et al, 2001; Hauschildt S et al, 1990) NO˙ can also come directly from dietary nitrates and nitrite (Lundberg JO et al, 2009; McKnight

GM et al, 1997 & 1998) Nitrite (NO2-) is the inert oxidative breakdown product of endogenous NO˙ It can be recycled back to bioactive NO˙ in blood and tissue and thus NO2˙-is thought to serve as part of NO˙ storage system in biological systems (Lundberg JO and Weitzberg E, 2005 & 2010) NO˙ storage system consists of free NO˙, NO2˙- and NO˙ adducts such as GSNO, protein-SNO and protein-bound dinitrosyl iron complexes The most important NO˙ storage protein is S-nitroso-haemoglobin (Hb-SNO) that travels throughout the body subserving NO˙ homeostasis (Angelo M et al, 2008; Martínez MC and Andriantsitohaina R, 2009; Muller B et al, 2002)

The most important NO˙ derived reactive nitrogen species is peroxynitrite (ONOO-) which is formed by the reaction of NO˙ with O2˙- at diffusion-controlled rates (∼ 1X1010 M-1 S-1) (Pacher P et al, 2007) The oxidant reactivity of ONOO- is highly pH-dependent and at physiological pH (pH 7.4), it has a very short half-life (∼10ms) ONOO- or its protonated form, peroxynitrous acid (ONOOH) reacts directly with various biomolecules or undergoes decomposition giving rise to peroxynitrite-derived secondary radicals The biochemistry of RNS is a complex process involving decomposition and recombination of individual RNS (Table 3) and occasionally involves bidirectional transition from one form to another For example, dinitrogen trioxide N2O3 derived from reaction of NO˙ with molecular oxygen, O2 is a powerful S-nitrosylating agent resulting in formation of S-nitrosothiols (RSNOs) or S-nitrosylated proteins (Grisham MB et al, 1999; Stamler JS and Hausladen A, 1998) Upon degradation, RSNOs in turn release NO˙ back to the NO˙ pool (Benhar M et al, 2009) Similarly, once considered as a final metabolite of NO˙ together with NO3-,

NO2- revert back to NO˙ particularly under hypoxic conditions by the action of xanthine oxidase (Martínez MC and Andriantsitohaina R, 2009) Decomposition of ONOO- gives rise to various RNS such as NO2-, NO2+, N2O3 and NO2 ˙ (Martínez MC and Andriantsitohaina R, 2009; Pacher P et al, 2007) (See chapter 4 for more details)

Table 3: Major Reactive Nitrogen Species in Biological System

(Adapted from Martínez MC and Andriantsitohaina R, 2009)

1.3 EFFECTS OF REACTIVE OXYGEN AND NITROGEN SPECIES ON

CELLULAR STRACTURE AND SIGNALING

In contrast, H2O2 reacts directly with transition metal centre of some heme-containing enzymes and thiolate of protein sulfhydryl (Forman HJ, 2004) OH˙ has a very high reactivity, with a very short half-life of approximately 10-9 s It reacts indiscriminately with biomolecules close to its site of formation (D'Autréaux B and Toledano MB, 2007; Riley, 1994) Toxicity associated with ROS in general and H2O2 in particular is mainly accounted for by excessive generation of OH˙ (Koppenol WH, 2001) OH˙ reacts with all components of the DNA molecule, damaging both purine and pyrimidine bases and the deoxyribose backbone (Halliwell B and Gutteridge JMC, 2007) The formation of highly mutagenic oxidative product 8-OH-G by the hydroxyl radical represents the initial step in free radical induced carcinogenesis (Valko M et

al, 2004; Wiseman H and Halliwell B, 1996) Alternatively, OH˙ can initiate a free radical chain reaction called “lipid peroxidation” by reacting with polyunsaturated fatty acid in the cell membrane with the final product being malondialdehyde (MDA) (Marnett LJ, 2002; Wang M et al, 1996) MDA reacts with nucleoside bases of DNA

to form highly mutagenic adducts such as M1G, M1A and M1C (Wang M et al, 1996) Oxidation of proteins by ROS can be reversible or irreversible depending on the

target, concentration of the oxidants and the type of oxidative damage Amino acids targets that are highly suspectible to oxidation include cysteine, methionine, lysine, arginine, threonine and histidine and proline The hallmark of oxidative damage to the proteins is formation of protein carbonyls, and as a result, the protein carbonyls content (PCC) is used as a measure of protein oxidation caused by oxidative stress (Dean RT et al, 1997; Valko M et al, 2006) Generally, cells can remove irreversibly oxidized proteins by proteolysis but when the amount of damaged protein exceed the cellular capacity to handle, pathologies such as diabetes, atherosclerosis and neurodegenerative diseases result (Davies KJ, 2001; Dean RT et al, 1997) The toxicity of peroxynitrite occurs through two mechanisms: oxidation and nitration resulting in altered protein function, apoptotic cell death and even tissue necrosis depending on the concentration and duration of exposure (Ahmad R et al, 2009; Szabó C 1996 & 2003) In the body, immune cells employ the toxicity of these ROS/RNS to launch a defense against invading microorganisms in innate immunity Although the production of ROS/RNS in this context is highly regulated and localized, sustained ROS/RNS producing activities of these cells are associated with various chronic inflammatory conditions and cancer (Karin M et al, 2006; Kohchi C

et al, 2009; Lambeth JD, 2007)

1.3.2 Physiological Function: Redox Signaling

While free radicals are traditionally linked to cell damage and death, recent compelling data indicates that they also play a major role in several aspects of signal transduction ranging from cell proliferation, migration, contraction to secretory function (D'Autréaux B and Toledano MB, 2007; Giles GI, 2006; Hool LC, 2006;; Trachootham D et al, 2008) This aspect of signal transduction is often referred to as

“Redox Signaling” It is established that cells are capable of generating low concentrations of ROS when stimulated by various ligands such as cytokines, growth factors and hormones (Petry A et al, 2010) Intentional generation of ROS was first observed in immune cells such as neutrophils and macrophages but certain cytokines such as TNF-α, IL-1 and interferon (IFN-γ) were later found to produce ROS in non-phagocytic cells as well (Matsubara Tvand Ziff M, 1986; Meier B et al, 1989; Yang D

et al, 2007) This was followed by reports of ROS generation upon growth factors binding to their receptors, and ROS thereby produced are required for their various functions (Bae YS et al, 1997; Marumo T et al, 1997; Shibanuma M et al, 1991; Sundaresan M et al, 1995) Thannickal VJ and Fanburg BL have compiled a list of ligands known to generate ROSs (Table 4) to which more members are being added and their functional significance is being unraveled

Table 4: List of Ligands inducing ROS production

(Adapted from Thannickal VJ and Fanburg BL, 2000)

ROS, reactive oxygen species; TNF-a, tumor necrosis factor-a; MCP-1, monocyte chemoattractant protein-1; CSF-1, colony-stimulating factor-1; NF-kB, nuclear factor-kB; IL, interleukin; HSP27, 27-kDa heat shock protein; COX-2, cyclooxygenase-2; IFN-γ, interferon- γ; PDGF, plateletderived growth factor; MAPK, mitogen-activated protein kinase; NOS, nitric oxide synthase; EGF, epidermal growth factor; JNK, c-Jun NH2-terminal kinase; PLA2, phospholipase A2; TGF, transforming growth factor; AOE, antioxidative enzyme; PLD, phospholipase D; 5-HT, 5-hydroxytryptamine (serotonin); ERK, extracellular signal-regulated kinase

ROS generated during signal transduction are now regarded as second messengers beacause they are generated at the time of receptor activation, are short-lived, and act specifically on effectors to transiently modify their activity (Sauer et al, 2001) Previously, there was skepticism about specificity of ROS to fit as signaling molecules because of highly reactive nature of ROS that renders them indiscriminate

in their reaction with biomolecules Conventionally, the specificity in cell signaling is achieved by virtue of complementarity of participating macromolecules that bind to each other non-covalently (Alberts B et al, 2008) But in ROS signaling, the specificity relies on the presence of susceptible targets and vicinity of targets to the site of ROS/RNS production (D'Autréaux B and Toledano MB, 2007) Proteins vary widely in their oxidant sensitivity and only a small number of highly sensitive proteins are suitable for redox signaling The redox sensivity of a particular protein is determined by the presence of oxidizable amino acids The most important amino acid

is cysteine that contains sulfhydryl or thiol (-SH) Its thiolate form (-S-) is far more nucleophilic than its protonated counterpart making it suitable for target of oxidation (Cross JV and Templeton DJ, 2006; Paulsen CE and Carroll KS, 2010) The typical

pKa of cysteine residue is 8.5 implying that there would be < 10% in the thiolate form

at a physiological pH of 7.2, but in the case of the susceptible proteins in which the

pKa can be lowered to 4-5, the proportion of deprotonated cysteine rises up to >50%

In other words, the cysteine is rendered “reactive”(Winterbourn CC and Hampton

MB, 2008) The low pKa is achieved through electrostatic interactions of neighboring basic and acidic residues, which are either present in the primary structure or higher order configurations (Buchner J and Moroder L, 2009) Thus, in a single protein, only very few cysteines out of several present is targeted by oxidation A case in point is the ryanodine receptor that contains approximately 50 free thiols in homotetrameric RyR monomer but only one single cysteine Cys3635 was identified as being redox-active (Eu JP et al, 2000).Although methionine, tryptophan, and tyrosine residues are reported to be susceptible to oxidation, the physiological significance of these events

is not fully understood

SIGNALING: FUNCTIONAL CONSEQUENCES OF OXIDATION OF “REACTIVE CYSTEINE”

The notion of ROS/RNS acting as signaling molecules comes from evidence that reaction of these oxidants with signaling proteins results in alteration of protein functions (Janssen-Heininger YM et al, 2008) Majority of the targets proteins by oxidation includes phosphatases, kinases, ion channels, chaperone proteins and transcription factors Function of these proteins is modified as a result of oxidation of reactive cysteine(s) within the proteins The summary of possible functional consequences by cysteine oxidation is depicted in figure 2 and details are given in the respective sections that follow

(Taken from Janssen-Heininger YM et al, 2008)

Figure 2: Consequences of oxidation of reactive cysteines within the target proteins

Consequences of protein oxidation are varied Activities of some proteins are inhibited by

cysteine oxidation whereas others are activated upon redox modification Likewise,

multimerization as a result of oxidation could lead to either inhibition or activation

Transcription factors can be activated by oxidizing their negative regulators or by direct

oxidation of their redox active cysteine residues ( -SH, reduced cysteine; -S-Ox, oxidized

cysteine)

1.4.1 Inhibition of Activity

Functional inactivation of proteins by oxidation is best characterized for protein tyrosine phosphatases (PTPs) and caspases (Denu TM and Tanner KG, 1998; Haendeler J et al, 1997; Li J et al, 1997) All PTPs contain a cysteine residue in their signature motif, CX5R The unique environment surrounding this active site motif lowers the pKa of the cysteine to ∼5.4 (Lohse DJ et al, 1997) This thiolate anion participates in the formation of a thiol-phosphate intermediate that is essential for catalytic mechanims of PTPs (Zhang ZY, 2003) Oxidation of the active-site cysteine

residue results in inhibition of enzyme activity and an increase in tyrosine phosphorylation (Chiarugi P and Cirri P, 2003; Cho SH et al, 2004; Tanner JJ et al, 2011) Similarly, proapoptotic proteins, caspases contain a cysteine in their active site The reduced state of the cysteine is required for the enzymatic activities of caspases thus making them susceptible to oxidative inactivation (Mannick JB, 2007; Tenneti L

et al, 1997) On one hand, s-nitrosylation (a form of oxidative modification, see below) of caspases under basal condition is associated with inhibition of their activities and prevention of apoptosis (Li J et al, 1997) but on the other hand, denitrosylation (reducing) of caspases following a variety of apoptotic stimuli triggers apoptosis (Kim TE and Tannenbaum SR, 2004; Mannick JB, 1999) Recently, it was suggested that antiapoptotic effect of the antioxidant protein, thioredoxin 1, Trx1 might exert through transnitrosylation of caspase-3 (Mitchell DA and Marletta MA 2005; Mitchell DA et al, 2007)

1.4.2 Activation of Protein Functions

Some kinases and ion channels are activated as a result of oxidation of their reactive cysteines Activation of many small GTPases within the Ras superfamily by S-nitrosylation is implicated in Angiotensin II signaling, the adaptive immune response and tumor maintenance (Heo J and Campbell SL, 2004; Raines KW et al, 2007) N-Ras is S-nitrosylated at cysteine 118 in eNOS dependent manner in T-cells Mutation of this redox sensitive cysteine residue leads to abrogation of Angiotensin II signaling and T-cells response mediated by N-Ras activation (Ibiza S et al, 2008) In skeletal muscles, high-conductance Ca2+ release channels or ryanodine receptors (RyR) are activated by various ROS/RNS signals (Anzai K et al, 2000) Out of 50 free cysteine residues in RyR, Cys 3635 was identified as the site of S-nitrosylation by

NO˙ that modulate channel activity (Sun J et al, 2001) In general, redox activation

of enzymes and channels occurs in an allosteric manner rather than direct impact on the active sites (Klomsiri C et al, 2011)

1.4.3 Multimerization of Subunits

Another mechanism by which cysteine oxidation influences the signal transduction is through multimerization For example, Hsp33 in its inactive state has four reactive cysteine residues in the thiolate anion form that bind Zn2+ Upon oxidation, each monomer releases the bound Zn2+ to form a homodimer, which enables the full activation of Hsp33’s function as a chaperone (Jakob U et al, 1999 & 2000; Winter J et al, 2005) The other chaperone proteins that are subject to oxidative dimerization and activation include mammalian Hsp25, 60, 70 and 90 (Diaz-Latoud C

et al, 2005; Hoppe G et al, 2004; Nardai G et al, 2000) On the other hand, formation

of Src protein tyrosine kinase homodimers through a disulphide bond between Cys277 of two monomers upon oxidation results in inactivation of the protein (Sun G and Kemble DJ, 2009)

1.4.4 Release of Regulatory Proteins

Redox regulation of signaling is also achieved by oxidation of a regulatory protein, which then dissociates from its binding partner, thereby activating the function of the partner The prime example of this is the redox regulation of ASK-1 function by Trx1 (Saitoh M et al, 1998) In the reduced state, Trx1 binds ASK-1 keeping the activity of ASK-1 in check Formation of disulphide linkage between the reactive cysteines 32 and 35 of Trx1 as a result of oxidation releases Trx1 from ASK-

1, which results in the activation of ASK-1 ASK-1 is a known activator of JNK that triggers cell death This mechanism of redox dependent regulation of cell death by Trx1 has been demonstrated in macrophages that were stimulated to produce H2O2

(Liu H et al, 2006) Another example is Nrf2-KEAP regulatory system in response to oxidants Nrf2 is a transcription factor that activates antioxidant responsive element-dependent gene expression to maintain redox homeostasis (Itoh K et al, 2004; Zhang

DD, 2006) Under normal conditions, KEAP-1 binds to Nrf2 and Cul3-based ligase promoting rapid degradation of Nrf2 via ubiquitin proteasome system Upon oxidative stress, oxidation of cysteines 273 and 288 in KEAP-1 causes its dissociation from Nrf2 allowing accumulation of Nrf2 in the nucleus and activation of target genes (Dinkova-Kostova AT et al, 2002; Kobayashi A et al, 2004; Tong KI et al, 2006; Wakabayashi N et al, 2004)

1.4.5 Oxidation of Transcription Factors

The transcription factors themselves are the direct targets of oxidation modulating their activities That was first demonstrated in the bacterial transcription factor OxyR which acts as a redox sensor, particularly to H2O2 Oxidation of Cys199 causes conformational change in dimeric OxyR, which then triggers the activation of OxyR site-specific binding to DNA promoters (Choi H et al, 2001; Christman MF et

al, 1985; Lee C at al, 2004) Similarly, oxidation of Cys-800 of HIF-1 alpha by nitrosylation stimulates its transcriptional activity (Sumbayev BB et al, 2003; Yasinska I et al, 2003) On the other hand, the reduced form of Cys62 of the subunit p50 is essential for the DNA binding capacity of NF-KB, a transcription factor for genes involved in inflammatory response, growth and differentiation (Marshall HE and Stamler JS, 2001; Pineda-Molina E et al, 2001) Similarly, the transcription factor

S-AP-1, the critical cysteine residues present in the DNA binding domain such as Cys

154 of c-Fos subunit and Cys 269 and 320 of c-Jun unit must remain reduced for DNA binding at tetradecanoylphorbol acetate (TPA) response element, TRE (Klatt P

at al, 1999; Hsu TC et al, 2000)

One feature rendering specificity to ROS/RNS mediated signal transduction is selective chemical reactivity of cysteine residues within the proteins Another essential feature in redox signaling is reversibility of protein oxidations Highly reactive species such as OH˙, hypochlorous acid, lipid peroxides and many decomposition products of ONOO- lack specificity, and most oxidation caused by these oxidants are not easily reduced and thus unlikely to participate in signal transduction For example, 3-nitrotyrosine and protein carbonyls caused by peroxynitrite are not reducible and their presence is associated with aberration of signal transduction and various pathologies (Beal MF, 2002; Ischiropoulos H and al-Mehdi AB, 1995) In contrast, Cys-based modifications have emerged as possible physiologically relevant redox-based post-translational protein modifications because some types of oxidations occurring to the cysteines are reversible by either intracellular reductants or enzymes Five types of reversible modification of reactive cysteines (S-) have been recognized and they are depicted below together with enzyme systems controlling respective cysteine oxidations

(Adapted Modified from Janssen-Heininger YM et al, 2008)

Figure 3: Schematic representation of various types of reversible cysteine oxidations

S- reactive cysteine, S-S disulphide, PSSG glutathionylation, SNO nitrosylation, SOH sulphenic acid, SO2H sulfinic acid

Table 5: Enzymes that reduce reversible cysteine oxidation

(Taken from Janssen-Heininger YM et al, 2008)

Reversible oxidation of the reactive cysteine was initially studied following

H2O2 mediated oxidation Oxidation by H2O2 produces a variety of reaction products (Stone JR and Yang S, 2006) such as sulphenic acid (SOH) which is highly unstable and go on to form sulfinic acid or disulphide bond (S-S) if there is a neighbouring reactive cysteine within the molecule (intramolecularly) or outside the molecule (intermolecularly) (Denu JM and Tanner JE, 1998; Salmeen A et al, 2003; Sarma BK

and Mugesh G, 2007) S-nitrosylation (SNO) is the covalent addition of NO group to

a reactive cysteine mediated by various S-nitrosylating species derived from nitric oxide (Hess DT et al, 2005) while s-glutathionylation (PSSG) is the addition of gluthione (GSH), or other low-molecular-weight thiols to the cysteine sulfhydryl and

it usually occurs under oxidative stress (Gallogly MM and Mieyal JJ, 2007)

CONSEQUENCES

The functional consequences of protein oxidation do not always follow a simple “On-Off” switch mechanism With more mechanistic insight into oxidative modification of proteins acquired, fine-tuning nature of redox regulation of protein functions become clear Generally, there are three peculiar features of cysteine oxidation Firstly, a single cysteine residue could be subject to alternative redox-based modifications resulting in differential functional outcomes The archetypical example

of this is a redox regulatory protein, OxyR OxyR is a DNA binding protein and transcription factor that induces multiple genes in response to oxidative and nitrosative stress Various groups have reported that the activity of OxyR is controlled

by a variety of redox-modifications at Cys199 (Choi H et al, 2001; Hausladen A et al, 1996; Zheng M et al, 1998;) Stimulants inducing oxidative, nitrosative or glutathionylation stress lead to formation of Cys199-SOH, Cys199-SNO, Cys199-SSG respectively with differences in conformational change, co-operative properties, DNA binding abilities and promoter activities of OxyR This is believed to underlie the differential and graded transcriptional responses observed with specific physiological stimulants (Kim SO et al, 2002) Secondly, depending on redox modification, the same protein could perform different or sometimes totally opposite

functions Trx1 is cytosolic protein of Trx family In the reduced state, it binds to and

inhibits ASK1, an activator of the c-Jun N-terminal kinase (JNK) and p38 MAPK

kinase pathway Intramolecular disulphide bond formation between Cys 32 and Cys

35 under oxidative stress results in dissociation of Trx1 from ASK-1 leading to activation of ASK-1 and ultimately cell death (Liu H et al, 2000 & 2006; Saitoh M et

al, 1998) On the other hand, cysteine 69 of Trx1 is S-nitrosylated under basal condition This is required for its oxidoreductase activity for scavenging of reactive oxygen species and protection from apopotosis (Haendeler A et al, 2002) Furthermore, S-nitrosylated Trx1 specifically transnitrosylates procaspase 3, thus inhibiting the activity of the latter and protecting cells from apoptosis (Mitchell DA and Marletta MA 2005; Mitchell DA et al, 2007) Lastly, the reactive cysteines of proteins could vary in their thresholds to oxidative modification with some displaying ready oxidation at lower threshold than others Numajiri and co-workers recently reported that at low concentration of NO˙, only PTEN is S-nitrosylated and inactivated, and as a result, Akt is phosphorylated whereas at high NO˙ concentration, both PTEN and Akt are S-nitrosylated leading to inactivation of Akt (Numajiri N et